The present invention relates to compounds having protein Kinase C inhibitory activity. More particularly, the present invention relates to alkyloxyamino-substituted fluorenones which inhibit protein kinase C, pharmaceutical compositions including these compounds and methods of using these compounds to control protein kinase C activity in mammals, including humans. More specifically, the present compounds are useful for the treatment of neoplastic disease states, disorders associated with abnormal blood flow (including hypertension, ischemia, atherosclerosis, coaggulation disorders), and inflammatory diseases (including immune disorders, asthma, lung fibrosis, psoriasis).
Protein kinase C (PKC) constitutes a family of enzymes called protein kinases. Activation of a protein kinase catalyzes the transfer of the .gamma.-phosphate of MqATP to either a serine, threonine residue of a particular protein substrate. Protein kinases regulate virtually all cell functions through the phosphorylation of proteins that are responsible for signal transduction and the regulation of cellular processes. Inhibitors of protein kinases could thus alter cell activities and thus can serve as pharmacological agents. Protein kinase C is a phospholipid-dependent, calcium activated serine/threonine protein kinase which was first characterized in the brain by Y. Nishizuka, Science 233:305-312 (1986). Subsequently, PKC was further characterized as a family of at least 11 enzymes. A. Azzi et al., Eur.J.Biochem. 208:547-557 (1992); D. S. Lester and R. M. Epand, Current Concepts and Future Perspectives, Ellis Horward, New York (1992); H. Hug and T. F. Sarre, Biochem. J. 291:329-343 (1993); C. Tanaka and Y. Nishizuka, Annu. Rev. Neurosci. 17:551-67 (1994); S. E. Wilkinson, T. J. Hallam, Trends Pharm. Sci. 15:53-57 (1994); E.O Harrington and A. J. Ware, Trends Cardiovas. Med. 5:193-199 (1995); A. C. Newton, J. Biol. Chem. 270:28495-28498 (1995); all of which are herein incorporated by reference.
Members of the Protein Kinase C family consist of a single polypeptide with a N-terminal regulatory end and a C-terminal catalytic end. The regulatory domain for all of the isozymes requires phospholipid for enzymatic activity, but only some of them require calcium for activation and some of them are not activated by phorbol myristate acetate (PMA), and agent long associated with PKC activation. On the other hand, the catalytic portion of the PKC enzymes are quite similar, but they share limited homology with other classes of kinases. For example, there is only 40% homology in this region with another well characterized protein kinase, protein kinase A (PKA). Analysis, of the catalytic site also uncovered a cluster of acidic residues unique to the protein kinase C family of enzymes J. W. Orr, A. C. Newton, J. Biol. Chem. 269:8383-8367 (1994). Thus, inhibitors or pharmaceutical agents directed at the catalytic site of this enzyme would be expected to be specific for the protein kinase C family of enzymes. Because of the potential therapeutic value of inhibitors of PKC, there is considerable effort being made to identify such substances. H. H Grunick, F. Ueberall, Sem. in Cancer Biol. 3:351-360 (1992); D. Bradshaw et al., Agents Actions 38:135-147 (1993); W. Harris et al., Drugs Future 18:727-735 (1993); A. Levitzi, Eur. J. Biochem. 226:1-13 (1994); K. J. Murray and W. J. Coates, Annu. Reports Med. Chem. 29:255-264 (1994); P. C. Gordge and W. J. Ryves, Cell. Signal 6:871-882 (1994); P. M. Blumberg et al., Agents Action Suppl. 47:87-100 (1995); J. C. Lee and J. L. Adams, Curr. Opp. Biotech. 6:657-661 (1995); all of which are herein incorporated by reference.
PKC regulates key steps in cell proliferation and cell growth. Potent activators of PKC, such as the phorbol esters, are well-known carcinogens. Thus, inhibitors of PKC would also be expected to be anticancer agents. The anticancer activity of several PKC inhibitors is well known, Grunick, supra., Levitzi, supra., Lee et al., supra, T. Meyer et al., Int. J. Cancer 43:851-856 (1989); S. Akinagaka et al., Cancer Res. 51:4888-4992 (1991), including activity against tumors expressing the multi-drug resistance phenotype I. Utz et al, Int. J. Cancer 57:104-110 (1994); all of which are herein incorporated by reference. A PKC inhibitor showed specificity in suppressing CDC2 kinase activity and in arresting cell cycling J. Hoffman et al., Biochem. Biophys. Res. Commun. 199:937-943 (1994), herein incorporated by reference. Inhibition of protein kinase C has been demonstrated to suppress tumor metastasis, J. A. Dumont et al., Cancer Res. 52:1195-1200 (1992), J. A. Dumont et al., Biochem. Biophys. Res. Comm. 204:264-272 (1994); all of which are herein incorporated by reference. Angiogenesis, the formation of new blood vessels, when associated with tumor formation and growth, fosters such formation and growth and in such instances is a pathological event.
Protein kinase C activation has been linked to mechanisms that set the rate of flow in blood vessels. Excessive PKC activity is likely implicated in hypertension, ischemia, and athersclerosis; all of these processes contribute to impaired blood flow, and can lead to ischemic heart disease, myocardial infarction, or stroke. Phorbol esters will produce increased contractility in the arteries from hypertensive rats, along with elevated PKC activity intrinsic to their vasculature and platelets, E. O. Harrington and A. J. Ware, supra; D. Bradshaw, et al., supra. Platelet aggregation is accompanied by PKC activation, contributing to abnormal clot formation and blood flow obstruction. M. A. Evans, et al., Br. Heart J. 68:109, herein incorporated by reference. Hyperplasia and proliferation of vascular smooth muscle plays a central role in the formation of atherosclerotic plaques. Activation of PKC is involved in smooth muscle proliferation E. O. Harrington and A. J. Ware, supra.
Tissues and organs become inflamed as a result of infiltration by certain activated white blood cells. The long term consequences can be tissue damage resulting in fibrosis. Both inflammatory cell activation and fibrotic processes have been associated with the excessive activity of protein kinase C. The T lymphocyte (T-cell) is the coordinator of immune responses. PKC plays a central role in the activation of these cells and PKC inhibitors will inhibit their activation and growth. D. Bradshaw et al., supra, W. Harris et al., supra, S. S. Alkkan et al., Cell. Immunol. 150:137-148 (1993), herein incorporated by reference. Not surprisingly, PKC inhibitors have been demonstrated to have activity in diseases where T-cells play a major role, such as transplant rejection J. P. Demers et al, Bioorg. Med. Chem. Lett. 4:2451-56, and psoriasis. L. Hegemann et al., Arch. Dermatol. Res. 284:179-183 (1992); J. J. Tegeler et al., Bioorg. Med. Chem. Lett. 5:2477-2482 (1995); all of which are herein incorporated by reference. White blood cells, such as monocytes (macrophages), neutrophils, and eosinophils can be activated by phorbol esters and their activities, such as free radical generation, phagocytosis, chemotaxis, and secretion can be suppressed by inhibitors of PKC. C-K Huang and R. I. Sha'afi, Protein kinases in blood cell function, CRC press, Boca Raton, Ch. 3,4,5; herein incorporated by reference. These granulocytes participate in and are largely responsible for the acute inflammatory processes that occur in such diseases as asthma, allergies and gouty arthritis. Thus, it is expected that inhibitors of PKC would have general antiinflammatory activity. A consequence of chronic inflammation is fibrosis. It is particularly notable that the class of compounds (fluorenones) described in this patent, and which we have identified as inhibitors of PKC, suppress alveolar macrophage activation and fibroblast proliferation and thus would be expected to have antiinflammatory and anti-fibrotic activity in the lung, J. Y. C. Ma et al., Exp. Lung Res. 21:771-790 (1995); herein incorporated by reference.
As related above, the implication of PKC in several human disease processes, including neoplastic disease states, vascular perfusion disorders, and inflammation, the inhibition of this enzyme would be expected to be of great value in treating these disorders. Furthermore, PKC inhibitors which are highly specific for the PKC class of protein kinases, which have minimal effects on other metabolic pathways such as those associated with stimulation of protein kinase A by cAMP are greatly desired. Neoplastic disease states, vascular perfusion disorders, and inflammation are common conditions for which there still exists a great need for novel and more definitive treatments.